Forkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such
as Muscle RING finger 1 (MuRF1) are believed to participate in the
regulation of skeletal muscle mass. The function of FoxO transcription
factors is regulated by post-translational modifications such as
phosphorylation and acetylation. In the present study FoxO1 protein
expression, phosphorylation and acetylation as well as MuRF1 protein
expression, were examined in atrophic and hypertrophic denervated
skeletal muscle.

Methods

Protein expression, phosphorylation and acetylation were studied
semi-quantitatively using Western blots. Muscles studied were 6-days
denervated mouse hind-limb muscles (anterior tibial as well as pooled
gastrocnemius and soleus muscles, all atrophic), 6-days denervated mouse
hemidiaphragm muscles (hypertrophic) and innervated control muscles.
Total muscle homogenates were used as well as separated nuclear and
cytosolic fractions of innervated and 6-days denervated anterior tibial
and hemidiaphragm muscles.

Results

Expression of FoxO1 and MuRF1 proteins increased 0.3-3.7-fold in all
6-days denervated muscles studied, atrophic as well as hypertrophic.
Phosphorylation of FoxO1 at S256 increased about 0.8-1-fold after
denervation in pooled gastrocnemius and soleus muscles and in
hemidiaphragm but not in unfractionated anterior tibial muscle. A small
(0.2-fold) but statistically significant increase in FoxO1
phosphorylation was, however, observed in cytosolic fractions of
denervated anterior tibial muscle. A statistically significant increase
in FoxO1 acetylation (0.8-fold) was observed only in denervated anterior
tibial muscle. Increases in total FoxO1 and in phosphorylated FoxO1 were
only seen in cytosolic fractions of denervated atrophic anterior tibial
muscle whereas in denervated hypertrophic hemidiaphragm both total FoxO1
and phosphorylated FoxO1 increased in cytosolic as well as in nuclear
fractions. MuRF1 protein expression increased in cytosolic as well as in
nuclear fractions of both denervated atrophic anterior tibial muscle and
denervated hypertrophic hemidiaphragm muscle.

Conclusions

Increased expression of FoxO1 and MuRF1 in denervated muscles (atrophic
as well as hypertrophic) suggests that these proteins participate in the
tissue remodelling occurring after denervation. The effect of
denervation on the level of phosphorylated and acetylated FoxO1 differed
in the muscles studied and may be related to differences in fiber type
composition of the muscles.

Skeletal muscle normally makes up about 45% of the body mass in humans [1] but is a very plastic tissue responsive
to alterations in usage. Muscle inactivity leads to a decrease in mass (atrophy)
whereas increased activity leads to an increase in mass (hypertrophy). Such
changes in muscle mass are believed to occur as a result of alterations in a
delicate balance between pathways regulating muscle protein synthesis and
degradation [2]. The Forkhead box O (FoxO)
transcription factors FoxO1 and FoxO3 are believed to participate in the
regulation of muscle mass since overexpression of these transcription factors
has been shown to lead to reduced skeletal muscle mass [3,4].

FoxO transcription factors include the four members FoxO1 (FKHR), FoxO3 (FKHRL1),
FoxO4 (AFX) and FoxO6 [5-7]. These are reported to have important
roles in e.g. stress resistance and metabolism by regulating the expression of
target genes. Examples of environmental stimuli that get translated by FoxO
transcription factors into specific gene expression programs include oxidative
stress, nutrients and growth factors [8].
In growing cells FoxO proteins are to a high extent located in the cytoplasm
[9] since nuclear export is a response
to growth signals and nuclear import is a response to stress signals such as
oxidative stress [9,10].

One effect of FoxO transcription factors that may be important for the regulation
of muscle mass is related to the control of transcription of E3 ubiquitin
ligases such as Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx,
Atrogin1). The mRNA expression of these ubiquitin ligases increase in a number
of different atrophic conditions, including immobilization, hind-limb
suspension, starvation, glucocorticoid treatment and denervation [11-17]. Similarly the mRNA expression of FoxO1 has been shown to
increase in a number of atrophic conditions including denervation [12,15,18]. Constitutively active
FoxO1, however, did not increase the expression of MAFbx or MuRF1 in myotubes
[19] and transgenic mice
overexpressing FoxO1 do not have consistent alterations in MAFbx or MuRF1 levels
[4]. FoxO1 has, however, been found to
cooperate with the glucocorticoid receptor to synergistically activate
transcription of a reporter gene driven by the MuRF1 promoter [20]. The nuclear content of FoxO1 protein
has been shown to decrease in human quadriceps muscle after resistance training,
associated with muscle growth, and then during a de-training period the amount
of FoxO1 protein increased in the nucleus [21].

The functions of FoxO transcription factors are controlled by post-translational
modifications such as phosphorylation, acetylation and ubiquitination that
influence transport between the nucleus and cytoplasm [22]. FoxO transcription factors can be phosphorylated by a
number of different kinases including Akt (protein kinase B). FoxO1 is
phosphorylated by Akt on S253, S316 and T24 (mouse FoxO1 sequence). The
phosphorylations occur sequentially starting with S253 in the forkhead domain
[9]. Following phosphorylation FoxO
transcription factors bind to 14-3-3 chaperone proteins and are transported out
of the nucleus to the cytoplasm. The 14-3-3 binding masks the nuclear
localization signal and this prevents FoxO from returning to the nucleus [10]. In C2C12 myotubes glucocorticoid
treatment or removal of growth medium has been shown to decrease the
phosphorylation of FoxO1 [3].

FoxO1 can also be acetylated at a number of different sites and acetylation seems
to have an inhibitory effect on DNA binding capability but may also stimulate
phosphorylation on S253 indicating that acetylation and phosphorylation may work
together to control the function of FoxO1 [7,23].

The purpose of the present study was to investigate FoxO1 protein expression,
phosphorylation and acetylation as well as MuRF1 protein expression in atrophic
(hind-limb) and hypertrophic (hemidiaphragm) 6-days denervated mouse skeletal
muscle. The hemidiaphragm muscle becomes transiently hypertrophic for
6–10 days following denervation [24-26] whereas hind-limb
muscles atrophy continuously following denervation. The hemidiaphragm of the
mouse contains mainly type II muscle fibers with a lower content (about 12%) of
type I fibers [27]. The hind-limb muscles
used in the present study were anterior tibial muscles that in the mouse are
devoid of type I muscle fibers [28] and
pooled gastrocnemius and soleus muscles that in addition to type II also contain
type I muscle fibers [28,29].

In innervated as well as in 6-days denervated atrophic anterior tibial muscle
total and phosphorylated FoxO1 protein were mainly present in cytosolic
fractions. Expression increased about 1-fold and 0.2-fold, respectively, in
cytoplasmic fractions of 6-days denervated muscles (Figure 6).

Figure 6

FoxO1 protein expression and phosphorylation levels in
cytosolic and nuclear fractions of 6-days denervated atrophic
anterior tibial muscle. Total FoxO1 protein expression
(A) and phosphorylation levels (B) in
cytosolic (C) and nuclear (N) fractions of 6-days denervated (Den)
atrophic anterior tibial muscle compared to innervated (Inn)
controls. Representative images of Western blots are shown together
with densitometric quantifications. One innervated cytosolic sample
was loaded onto all gels as a reference. All samples were measured
relative to this reference. The data were normalized so that the sum
of cytosolic and nuclear signals in innervated muscles will give a
mean value of 100.0. Mean values ± standard error
of the mean. Statistical comparisons were made between cytosolic
fractions of denervated versus innervated muscles and between
nuclear fractions of denervated versus innervated muscles.
**p < 0.01, n = 8 denervated anterior
tibial muscles and 8 contralateral innervated control muscles. Each
muscle was fractionated into a cytosolic and a nuclear fraction.

In innervated as well as in 6-days denervated hypertrophic hemidiaphragm
muscle total and phosphorylated FoxO1 protein were mainly present in
cytosolic fractions. Expression of total FoxO1 protein increased about
1.7-fold and 1.4-fold, respectively, in cytoplasmic and nuclear fractions of
6-days denervated muscles. Expression of phosphorylated FoxO1 increased
about 1.3-fold and 2.5-fold, respectively, in cytoplasmic and nuclear
fractions of 6-days denervated muscles (Figure 7).

Figure 7

FoxO1 protein expression and phosphorylation levels in
cytosolic and nuclear fractions of 6-days denervated
hypertrophic hemidiaphragm muscle. Total FoxO1 protein
expression (A) and phosphorylation levels
(B) in cytosolic (C) and nuclear (N) fractions of
6-days denervated (Den) hypertrophic hemidiaphragm muscle compared
to innervated (Inn) controls. Representative images of Western blots
are shown together with densitometric quantifications. One
innervated cytosolic sample was loaded onto all gels as a reference.
All samples were measured relative to this reference. The data were
normalized so that the sum of cytosolic and nuclear signals in
innervated muscles will give a mean value of 100.0. Mean
values ± standard error of the mean. Statistical
comparisons were made between cytosolic fractions of denervated
versus innervated muscles and between nuclear fractions of
denervated versus innervated muscles. *p < 0.05,
**p < 0.01, n = 8 denervated
hemidiaphragm muscles and 8 innervated control hemidiaphragms from
separate animals. Each muscle was fractionated into a cytosolic and
a nuclear fraction.

In innervated as well as in 6-days denervated atrophic anterior tibial muscle
and hypertrophic hemidiaphragm muscle MuRF1 protein was mainly present in
cytosolic fractions. Expression increased about 0.5-2.4-fold in cytoplasmic
as well as nuclear fractions of 6-days denervated muscles (Figure 8).

Figure 8

MuRF1 protein expression in cytosolic and nuclear fractions of
6-days denervated atrophic anterior tibial muscle and in 6-days
denervated hypertrophic hemidiaphragm muscle. MuRF1
expression in cytosolic (C) and nuclear (N) fractions of 6-days
denervated (Den) atrophic anterior tibial muscle (A)
and 6-days denervated hypertrophic hemidiaphragm muscle
(B) compared to innervated (Inn) controls.
Representative images of Western blots are shown together with
densitometric quantifications. One innervated cytosolic sample was
loaded onto all gels as a reference. All samples were measured
relative to this reference. The data were normalized so that the sum
of cytosolic and nuclear signals in innervated muscles will give a
mean value of 100.0. Mean values ± standard error
of the mean. Statistical comparisons were made between cytosolic
fractions of denervated versus innervated muscles and between
nuclear fractions of denervated versus innervated muscles.
*p < 0.05, ***p < 0.001,
n = 8 denervated hemidiaphragm muscles and 8
innervated control hemidiaphragms from separate animals. Each muscle
was fractionated into a cytosolic and a nuclear fraction.

The present study has examined the expression of FoxO1 protein and
post-translational modifications of FoxO1 in models of atrophic and hypertrophic
denervated skeletal muscle. Most denervated skeletal muscles atrophy but the
hemidiaphragm muscle undergoes a transient hypertrophy following denervation
possibly as a result of passive stretching due to continued contractions in the
contralateral innervated hemidiaphragm [24-26]. The hemidiaphragm of
the mouse is composed mainly of type II muscle fibers but also contains about
12% of type I fibers [27]. The hind-limb
muscles used in the present study were mouse anterior tibial muscles that are
devoid of type I muscle fibers [28] and
pooled gastrocnemius and soleus muscles that in addition to type II also contain
type I muscle fibers [28,29].

Similar to what has previously been shown for FoxO3 [30-32], and recently
also for FoxO1 in atrophic hind-limb muscle [33], the present study shows that the expression of FoxO1 protein is
increased in 6-days denervated skeletal muscle. This increase was observed in
all denervated muscles studied, atrophic as well as hypertrophic, suggesting
that FoxO1 plays a role for denervation changes other than those leading to
alterations in muscle mass. One such role may relate to the expression of
different myosin heavy chain isoforms. Thus, overexpression of FoxO1 has
previously been shown to result in a decrease in type I muscle fibers and a
strong reduction in the expression of the slow muscle myosin heavy chain isoform
[4]. Similarly, in soleus and
gastrocnemius muscles denervation has been shown to reduce the expression of the
slow muscle myosin heavy chain isoform [34,35]. Increased FoxO1
expression has also been reported in hypertrophic mouse plantaris muscle
following functional overload [36,37].

Phosphorylation of FoxO1 at S256 increased in pooled gastrocnemius and soleus
muscles (atrophic with type I fibers) as well as in hemidiaphragm (hypertrophic
with type I fibers) but not in unfractionated anterior tibial muscle (atrophic
without type I fibers). A small but statistically significant increase in FoxO1
phosphorylation was, however, observed in the cytosolic fraction of denervated
anterior tibial muscle. The difference in phosphorylated FoxO1 between
denervated anterior tibial and pooled gastrocnemius and soleus muscles might be
related to FoxO1 being more readily phosphorylated in type I muscle fibers as
suggested by a higher p-FoxO1/FoxO1 ratio in soleus muscle compared to anterior
tibial muscle [31]. A statistically
significant increase in FoxO1 acetylation was observed only in denervated
anterior tibial muscle. A previous study has reported increased acetylation of
FoxO3 in denervated anterior tibial muscle but at later times following
denervation [30].

FoxO1 protein expression and phosphorylation were also studied in separated
cytosolic and nuclear fractions of hemidiaphragm and anterior tibial muscles. In
all muscles studied, innervated as well as denervated atrophic and denervated
hypertrophic muscles, total and phosphorylated FoxO1 protein were mainly present
in cytosolic fractions. In anterior tibial muscle increases in protein
expression and phosphorylation were only observed in cytosolic fractions
following denervation. In hemidiaphragm total FoxO1 protein, as well as
phosphorylated protein, were increased in nuclear as well as in cytosolic
fractions following denervation. A previous study has also reported increased
nuclear FoxO1 in denervated rat hemidiaphragm although only at early times
(1 day) after denervation, but not after 5 days [38].

MuRF1 protein expression has previously been reported to increase in denervated
hind-limb muscle [33,39,40]. The present study confirms increased MuRF1 protein expression
in denervated atrophic hind-limb muscle but also shows that MuRF1 protein
expression is increased in denervated hemidiaphragm muscle at a time point when
the muscle is in a hypertrophic state relative to innervated control muscles.
Despite the hypertrophic state previous studies on denervated rat hemidiaphragm
indicate that from 5 days following denervation protein degradation, as
well as protein synthesis, is increased in this muscle [41]. Expression of MuRF1 has been reported to be controlled
by myogenin and deletion of myogenin diminishes the expression of MuRF1 in
denervated hind-limb muscles [42,43]. Increased expression of MuRF1 in
denervated muscle may therefore be a consequence of the increased expression of
myogenin that occurs following denervation in hind-limb as well as in
hemidiaphragm muscle [44-46]. MuRF1 has also been shown to be
preferentially expressed in type II muscle fibers, and also to be preferentially
induced in type II fibers after denervation [40,47]. All muscles included
in the present study contain type II muscle fibers but the fiber type
composition of muscles changes following denervation [48,49]. It is, thus,
also possible that the increased MuRF1 expression observed in the present study
relates to alterations in fiber types that occur following denervation.

Conclusions

Increased expression of FoxO1 and MuRF1 in denervated muscles (atrophic as well
as hypertrophic) suggests that these proteins participate in the tissue
remodelling that occurs in skeletal muscle following denervation. The effect of
denervation on the level of phosphorylated and acetylated FoxO1 differed in the
muscles studied and may be related to differences in fiber type composition of
the muscles.

MethodsAnimals and muscles

Adult male NMRI mice (Scanbur, Sollentuna, Sweden) were used in this study.
The mice were kept in cages with environment enrichment and with free access
to a standard laboratory diet and tap water. The animals were anaesthetized
by inhalation of isoflurane before surgery and received a subcutaneous
injection of buprenorphine (50 μg/kg) for post-operative
analgesia. Denervation of either the left hemidiaphragm or the left
hind-limb was performed by sectioning and removing a few mm of the phrenic
nerve or the sciatic nerve as described previously [50]. Six days after denervation the mice were killed by
cervical dislocation. Hind-limb muscles (anterior tibial and gastrocnemius
together with soleus) were rapidly dissected, weighed, frozen on dry ice and
stored at −80°C. Innervated control hind-limb muscles were
collected from the contralateral (right) leg of animals that were denervated
by sectioning the left sciatic nerve. Innervated left control hemidiaphragms
were collected from separate animals that had received no surgery. To
control for this, eight animals used for hemidiaphragm studies went through
sham surgery. These animals were anaesthetized by inhalation of isoflurane,
had a subcutaneous injection of buprenorphine (50 μg/kg) and a
unilateral thoracotomy without touching the phrenic nerve. For dissection of
the hemidiaphragm muscle the diaphragm, attached to the rib cage, was
quickly removed and placed in cold phosphate buffered saline (PBS) with
calcium (2 mM). The left hemidiaphragm was then dissected under a
dissecting microscope, blotted dry on filter paper, weighed, frozen on dry
ice and stored at −80°C. The experimental manipulations have
been approved by the Ethical Committee for Animal Experiments,
Linköping, Sweden (permit number: 67–10).

The expression levels of total, phosphorylated and acetylated proteins were
studied semi-quantitatively using data from Western blots. Equal amounts of
total, cytosolic or nuclear proteins from innervated or denervated muscles
were loaded on the gels. Measured levels of total, phosphorylated or
acetylated proteins are expressed without normalization to any specific
protein. No loading controls were used and any difference in protein
quantifications, pipettings steps, protein transfers etc. are included in
the variations of the data sets.

Image analysis was performed using the gel plotting macro of the program
ImageJ (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda,
MD, http://rsb.info.nih.gov/ij/, 1997–2007). Results were
obtained in uncalibrated optical density units

In order to be able to compare data for whole muscle homogenates run on
different gels, one innervated muscle sample (a reference sample) was
included in all gels containing samples to be compared to each other. All
other samples were measured relative to this reference, the signal of which
was set to 100.0 in all gels. In order to more easily compare denervated and
innervated muscles all data were finally normalized in such a way that the
average signal from innervated muscles became 100.0.

For quantification of protein expression in separated cytosolic and nuclear
fractions one of the cytosolic fractions from an innervated muscle was used
as a reference sample and was included in all gels. All other samples were
measured relative to this reference, the signal of which was set to 100.0.
From the amount of protein loaded on gels in relation to the total amount of
protein extracted in the nuclear and cytosolic fractions a total cytosolic
and a total nuclear signal was calculated for whole muscles. In the final
analysis total cytosolic and total nuclear signals were again normalized in
such way that the sum of the nuclear and cytosolic signals became 100.0 in
innervated muscle.

Data are presented as mean values ± standard error of the
mean (SEM). For statistical comparisons of unfractionated hemidiaphragm
muscles (innervated, sham operated and denervated) one way-ANOVA was used,
followed by Tukey’s multiple comparisons test, for normally
distributed data (according to D’Agostino-Pearson omnibus K2
normality test). Statistical significance for data not being normally
distributed was determined using the Kruskal-Wallis test with Dunn’s
multiple comparisons test. For other comparisons Student’s t-test
(paired observations for hind-limb muscles, unpaired observations for
hemidiaphragm muscles) was used for normally distributed data. Statistical
significance for data not being normally distributed was determined using
the Wilcoxon matched-pairs signed rank test (hind-limb muscles) or the
Mann–Whitney test (hemidiaphragm muscles).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

The work presented here was carried out in collaboration between all authors. AKF
designed the study and carried out most of the protein expression studies,
statistical analyses and drafted the manuscript. KE carried out the studies on
nuclear and cytosolic fractions and did the statistical analyses of these. MN
and ST conceived of the study, participated in the design, statistical analyses
and drafting of the manuscript. All authors have read and approved the final
manuscript.

Acknowledgements

We would like to thank Amanda Nyström for help with the MuRF1 studies. This
work was supported by grants from the Faculty of Health and Life Sciences,
Linnaeus University, Kalmar, Sweden.